Despite the pivotal role of Ca2+ homeostasis in myocyte physiology and disease, large gaps exist in our understanding of its compartmentalization within subcellular microdomains and its trafficking among them. A vast literature has glossed over the finely tuned handling of intracellular Ca2+, its sites of storage, release, action, and reuptake, focusing instead on crude measures of bulk Ca2+ concentration. In so doing, the intricacies of spatiotemporal handling of Ca2+, occurring on the scales of milliseconds and nanometers, are blurred, and our understanding is incomplete.

Appreciation of the pivotal role of Ca2+ in heart function dates to the late 19th century, when Sidney Ringer3 discovered that this cation is absolutely required for cardiac mechanical function. Cardiac contraction is triggered by influx of a small amount of Ca2+ through voltage-gated l-type Ca2+ channels (LTCC) embedded in the cell surface membrane. This Ca2+ influx, in turn, triggers the release of much larger amounts of Ca2+ from sarcoplasmic reticulum (SR) stores through ryanodine receptors (RyRs), a process termed Ca2+-induced Ca2+ release. Thus, LTCC Ca2+ influx gates Ca2+-induced Ca2+ release; in the converse sense, Ca2+ released by Ca2+-induced Ca2+ release feeds back to control LTCC influx.4 The end result is an elaborate, finely tuned, and self-regulated cascade of events that controls every heart beat.

The intricate dynamics of this process occur within, and are facilitated by, the microarchitecture of the junctional cleft, the region within the myocyte separating the SR and intramyocyte invaginations of the cell membrane (T-tubules).5 This zone, also termed the dyad, represents a volume of ≈10–3 fL in cardiac muscle,6 with ≈12 nm separating the LTCC within the T-tubule from the RyR where SR Ca2+ release takes place.7

Under steady-state conditions, Ca2+ entering through the LTCC is extruded back out of the cell by the Na+/Ca2+ exchanger.8 SR Ca2+ stores are replenished by the SERCA (sarco-endoplasmic reticulum Ca2+-ATPase) pump. Ca2+ concentrations in cellular microdomains and their time-dependent changes are also governed by Ca2+ buffering proteins, including calsequestrin and S100A1, and Ca2+ storage and release in mitochondria.

As complex as this picture is already, evidence is emerging that dyads differ in their structure and molecular architecture.9 In a variety of heart diseases, T-tubule and dyadic junction remodeling are seen, which likely participates in disease pathogenesis and distorted Ca2+ release.7 For all these reasons, it is of obvious importance to glean a more sophisticated understanding of Ca2+ handling within the junctional cleft, ideally with the spatial and temporal resolution required by the underlying biology.

Ca2+ Sensors

Using Ca2+-sensitive bioluminescent probes and fluorescent indicators, whole-cell Ca2+ concentration ([Ca2+]) during both systole and diastole has been studied extensively. The first experiments visualizing intracellular Ca2+ transients were reported in frog heart10 and canine Purkinje fibers11 using the Ca2+-sensitive bioluminescent protein aequorin. Because aequorin is membrane-impermeant, these and subsequent experiments were performed by injecting protein purified from the jellyfish Aequorea directly into cells. The associated technical challenges, coupled with modest signal-to-noise ratio afforded by the sensor, limited the utility of this approach.

Next, development of small-molecule chemical fluorescence indicators with high Ca2+ affinity and fast kinetics, such as Fura-2,12 and use of acetoxymethylated esters for noninvasive cell loading were breakthroughs that expanded the feasibility of Ca2+ imaging in single cardiomyocytes.13 Later, the advent of confocal microscopy and fast, high-contrast fluorescein- and rhodamine-based Ca2+ indicators, such as Fluo-3, led to the characterization of Ca2+ sparks, the result of single-dyad Ca2+ events.14

Whereas development of these diffusible indicators greatly facilitated our understanding of cardiac excitation–contraction coupling, they afforded neither the submicron spatial resolution nor the rapid on and off kinetics required for a complete understanding of this intricate Ca2+ biology. It is estimated that Ca2+ ions diffuse within myocytes at a rate of ≈100 μm/s15 and typically traverse a distance of ≈1.8 μm.16 Thus, conventional fluorescent Ca2+ indicators substantially underestimate local peak Ca2+ levels and report a low-resolution profile of local Ca2+ gradients.17

The next leap in Ca2+ imaging technology was introduction of genetically encoded Ca2+ indicators. Genetically encoded Ca2+ indicators offer advantages not achievable with conventional chemical Ca2+ probes, including precise targeting within cellular micro(nano)-compartments, prolonged imaging over days and months, and simultaneous multicolor Ca2+ imaging.18 Among the most popular protein calcium sensors are the GCaMPs, which are variants of enhanced green fluorescent protein (cpEGFP) coupled to a C-terminal Ca2+ sensor (calmodulin) and a calmodulin-binding M13 peptide at the N-terminus.19 On Ca2+ binding, the calmodulin moiety interacts with the M13 peptide, triggering structural reorganization in proximity to the cpEGFP, deprotonation of the chromophore, and increased fluorescence.20 GCaMPs are rapidly emerging as valuable tools for in situ monitoring of Ca2+ activity in neurons21 and heart.22

In this issue of Circulation Research, Despa et al23 describe 2 novel Ca2+ sensors specifically targeted to the junctional cleft. One harbors GCaMP2.2, a T203V mutation of GCaMP2 with intensified brightness and dynamic range.21 The second, GCaMP2.2Low, is a variant with reduced Ca2+ affinity, harboring a D133E mutation. Each construct was targeted to the dyadic cleft by means of covalent coupling to the N-terminus of FKBP12.6, a protein that binds with high affinity and selectivity to RyRs. Both fusion proteins retained high affinity for RyRs (Kd ≈15–45 nmol/L), albeit somewhat lower than native FKBP12.6 (1 nmol/L).24 A striated pattern of fluorescence, competed away by FKBP12.6, was observed in cultured adult rat ventricular cardiomyocytes, suggesting that both FKBP12.6-tagged sensors target selectively to the junctional cleft.

What Have These New Probes Taught Us?

GCaMP2.2Low manifests baseline fluorescence and a dynamic range similar to GCaMP2.2, but its affinity for Ca2+ is 10-fold lower (Kd, 5 μmol/L versus 450 nmol/L). These investigators capitalized on these differences to quantify the high [Ca2+] expected in the junctional cleft. Prior to this work, estimates of Ca2+ transient amplitudes near Ca2+ release sites ranged between 10 μmol/L8 to 100 μmol/L,25 and peak amplitude was estimated to occur 5 to 10 ms after depolarization, with recovery occurring within 15 to 50 ms.8,26 In contrast, the global Ca2+ transient within the cytoplasm is many fold lower, reaching a maximum of ≈1 μmol/L within ≈70 to 100 ms.8 Thus, GCaMP2.2Low, with its low Ca2+ affinity, could provide reliable measures of Ca2+ transients within the junctional cleft, whereas GCaMP2.2, with its high Ca2+ affinity, is optimal for measuring diastolic [Ca2+]i.

With these newly engineered probes in hand, these investigators first calibrated the targeted and untargeted sensors in the myocyte environment, which reduced affinities by ≈2-fold. However, FKBP12.6 targeting did not alter the Ca2+ affinity of either. Next, they set out to measure Ca2+ dynamics, reporting that [Ca2+]Cleft reaches higher levels (1.3 μmol/L) with faster kinetics than global [Ca2+]Bulk (0.5 μmol/L at peak). However, estimates of peak [Ca2+]Cleft reported with this sensor are much smaller than previous estimates, and the time to [Ca2+]Cleft peak (46 ms) is also much slower (5–10 ms). GCaMPs manifest significantly slower response kinetics (typically τon 20 ms to 1.4 seconds; τoff 0.4–5 seconds) compared with synthetic BAPTA-based dyes (eg, Oregon Green 488 BAPTA 5N; τon <1 ms).27 As acknowledged by the authors, the rapid rises and falls in [Ca2+]Cleft almost certainly exceed the kinetics of the GCaMP sensor. In this respect, it is highly likely that the low Ca2+ affinity, cleft-targeted GCaMP2.2Low significantly underestimated peak Ca2+ transients.

That being said, these novel sensors are ideally suited to measuring diastolic [Ca2+]Cleft. Because Ca2+ leak from the SR has been implicated in a variety of disorders, including heart failure,28 such measures are of great interest. To address this, the authors compared the signal emitted by GCaMP2.2 ([Ca2+]Bulk) with that emitted by GCaMP2.2-FKBP12.6 ([Ca2+]Cleft), noting that [Ca2+]Cleft was ≈2-fold higher than [Ca2+]Bulk (194 versus 100 μmol/L). Next, by selectively interrupting Ca2+ flux through LTCC versus RyRs, the authors went on to provide evidence that this resting diastolic gradient of Ca2+ stems from RyR leak, rather than sarcolemmal Ca2+ flux.

The Future Is Fast and Bright

Whereas these ingenious, cleft-targeted probes have provided novel insights already, challenges remain, especially given their slow kinetics. Recently, a new generation of fast GCaMPs was introduced.27 One of them, GCaMP6f, is reported to have fast kinetics (the fastest among all currently known GCaMPs), high affinity, and robust dynamic range; it was successfully implemented to measure single-neuron synapse events.29 Shang et al30 fused GCaMP6f with junctin or triadin and reported high-resolution images of junctional Ca2+ at individual dyads, so-called Ca2+ nanosparks. Nevertheless, GCaMP6f is still not fast enough to accurately track the extremely rapidly rising and fast-decaying Ca2+ gradient in the junctional cleft.31

Whereas GCaMP2.2Low–FKBP12.6 may not provide accurate measures of junctional cleft Ca2+ transients during excitation, GCaMP2.2–FKBP12.6 is an ideal sensor to measure [Ca2+]Cleft during diastole. Because heart failure and cardiac arrhythmias are often associated with dyssynchronous Ca2+ leak in diastole, this probe is likely to prove useful in dissecting underlying mechanisms. Also, high diastolic [Ca2+]Cleft may play an important role beyond governing excitation–contraction coupling by regulating local signaling cascades (eg, calcineurin or CaMKII).32 As a result, these clever new tools are likely to be informative in multiple domains of cardiovascular biology.

Sources of Funding

This work was supported by grants from the National Institutes of Health (HL-120732; HL-100401), American Heart Association (SDG18440002), Cancer Prevention Research Institute of Texas (RP110486P3), and the Leducq Foundation (11CVD04).

Disclosures

None.

Footnotes

The opinions expressed in this article are not necessarily those of the editors or of the American Heart Association.